Sterols are a class of essential, natural compounds required by all eukaryotes to complete their life cycle. The types of sterols produced and predominantly present within each of the phylogenetic kingdoms varies. Plants produce a class of sterols called phytosterols. A phytosterol called sitosterol predominates. In animals, cholesterol is typically the major sterol while in fungi it is ergosterol.
Phytosterols from plants possess a wide spectrum of biological activities in animals and humans. Phytosterols are considered efficacious cholesterol-lowering agents (Pelletier et al., Annals Nutrit. Metab. 39:291–295 (1995), the entirety of which is herein incorporated by reference). Lower cholesterol levels are linked to a reduction in the risk to cardiovascular disease. Phytosterols can also block cholesterol absorption in the intestine, which would also lead to lower cholesterol levels. Thus, enhancing the levels of phytosterols in edible plants and seeds, or products derived from these plants and seeds, may lead to food products with increased nutritive or therapeutic value.
In one aspect, this invention provides these desirable plants and seeds as well as methods to produce them. Since, as will be discussed below, the genetic manipulation made possible by this invention involves families of related genes that cross phylogenetic boundaries, the effects are not limited to plants alone.
Biochemistry of Sterol Synthesis
A number of the important sterol biosynthetic enzymes, reactions, and intermediates have been described. Sterol synthesis uses acetyl CoA as the basic carbon building block. Multiple acetyl CoA molecules form the five-carbon isoprene units, hence the name isoprenoid pathway. Enzymatic combination of isoprene units leads to the thirty-carbon squalene molecule, which is the penultimate precursor to sterols.
Throughout plants, animals, and fungus, the reactions proceed as: acetyl CoA_HMGCoA, mevalonate, mevalonate 5 phosphate, mevalonate 5-pyrophosphate, isopentyl diphosphate, 5-pyrophosphatemevalonate, isopentyl pyrophosphate (PIP), dimethylallyl pyrophosphate (DMAPP), PIP+DMAPP, geranyl pyrophosphate+IPP, farnesyl pyrophosphate, 2 farnesyl pyrophosphate, squalene and squalene epoxide
From squalene epoxide, the sterol biosynthesis pathway of plants diverges from that of animals and fungi. In plants, cycloartenol is produced next by cyclization of squalene epoxide. The plant pathway eventually leads to the synthesis of the predominant phytosterol, sitosterol.
Animals go on to produce lanosterol from squalene epoxide, eventually leading to cholesterol, which is the precursor to steroid hormones and bile acids, among other compounds. In fungi, lanosterol leads to the production of the predominant sterol, ergosterol.
An important regulatory control step within the pathway consists of the HMGCoA_Mevalonate step, catalyzed by HMGCoA reductase, and the condensation of 2 farnesyl pyrophosphates_squalene, catalyzed by squalene synthase. An early, reported rate-limiting step, in the pathway is the HMGCoA reductase-catalyzed reaction.
A number of studies have focused on the regulation of HMGCoA reductase. HMGCoA reductase (EC 1.1.1.34) catalyzes the reductive conversion of HMGCoA to mevalonic acid (MVA). This reaction is the controlling step in isoprenoid biosynthesis. The enzyme is regulated by feedback mechanisms and by a system of activation kinases and phosphatases (Gray, Adv. Bot. Res., 14: 25 (1987); Bach et al., Lipids, 26: 637 (1991); Stermer et al., J. Lipid Res., 35: 1133 (1994), all of which are herein incorporated by reference in their entirety).
Another important regulation occurs at the squalene synthase step. Squalene synthase (EC 2.5.1.21) reductively condenses two molecules of FPP in the presence of Mg2+ and NADPH to form squalene. The reaction involves a head-to-head condensation and forms a stable intermediate, presqualene diphosphate. The enzyme is subject to regulation similar to that of HMGCoA reductase and acts by balancing the incorporation of FPP into sterols and other compounds.
The sterol pathway of plants diverges from that in animals and fungi after squalene epoxide. In plants, the cyclization of squalene epoxide occurs next, under the regulated control of cycloartenol synthase (EC 5.4.99.8). The cyclization mechanism proceeds from the epoxy end into a chair-boat-chair-boat sequence that is mediated by a transient C-20 carbocationic intermediate. The reported rate-limiting step in plant sterol synthesis occurs in the next step, S-adenosyl-L-methionine:sterol C-24 methyl transferase (EC 2.1.1.41) (SMTI) catalyzing the transfer of a methyl group from a cofactor, S-adenosyl-L-methionine, to the C-24 center of the sterol side chain. This is the first of two methyl transfer reactions. The second methyl transfer reaction occurs further down in the pathway and has been reported to be catalyzed by SMTII. An isoform enzyme, SMTII, catalyzes the conversion of 24-methylene lophenol to 24-ethylidene lophenol (Fonteneau et al., Plant Sci Lett 10: 147–155(1977), the entirety of which is herein incorporated by reference). The presence of two distinct SMTs in plants were further confirmed by cloning cDNAs code the enzymes from Arabidopsis (Husselstein et al., FEBS Lett 381:87–92(1996), the entirety of which is herein incorporated by reference), soybean (Shi et al., J Biol Chem 271: 9384–9389(1996), the entirety of which is herein incorporated by reference), maize (Grebenok et al., Plant Mol Biol 34: 891–896(1997), the entirety of which is herein incorporated by reference) and tobacco (Bouvier-Nave et al., Eur J Biochem 246: 518–529 (1997); Bouvier-Nave et al., Eur J Biochem 256: 88–96(1998), both of which are herein incorporated by reference in their entirety).
Later in the pathway, a sterol C-14 demethylase catalyzes the demethylation at C-14, removing the methyl group and creating a double bond. Interestingly, this enzyme also occurs in plants and fungi, but at a different point in the pathway. Sterol C14-demethylation is mediated by a cytochrome P-450 complex. A large family of enzymes utilize the cytochrome P-450 complex. There is, in addition, a family of cytochrome P450 complexes. Sterol C-22 desaturase (EC 2.7.3.9) catalyzes the formation of the double bond at C-22 on the side chain. The C-22 desaturase in yeast, which is the final step in the biosynthesis of ergosterol, contains a cytochrome P450 that is distinct from the cytochrome P450 participating in the demethylation reaction. Additional cytochrome P450 enzymes participate in brassinosteroid synthesis (Bishop, Plant Cell 8:959–969 (1996), the entirety of which is herein incorporated by reference). Brassinosteroids are steroidal compounds with plant growth regulatory properties, including modulation of cell expansion and photomorphogenesis (Artecal, Plant Hormones, Physiology, Biochemistry and Molecular Biology ed. Davies, Kluwer Academic Publishers, Dordrecht, 66 (1995), Yakota, Trends in Plant Science 2:137–143 (1997), both of which are herein incorporated by reference in their entirety).
One class of proteins, oxysterol-binding proteins, have been reported in humans and yeast (Jiang et al., Yeast 10:341–353 (1994), the entirety of which is herein incorporated by reference). These proteins have been reported to modulate ergosterol levels in yeast (Jiang et al., Yeast 10: 341–353 (1994)). In particular, Jiang et al., reported three genes KES1, HES1 and OSH1, which encode proteins containing an oxysterol-binding region.
The present invention provides a gene, hes1, involved in plant phytosterol production. Expression of HES1 (protein) in organisms, such as plants, can increase phytosterol biosynthesis. The present invention also provides transgenic organisms expressing a HES1 protein, which can enhance food and feed sources.